As a pathogenic B cell response, factor VIII inhibitors represent a major clinical challenge including alloantibodies in hemophilia A patients or autoantibodies in acquired hemophilia. Responses are polyclonal to a limited number of major epitopes on different domains and IgG4 responses often predominate. However, most studies to characterize factor VIII inhibitors have been on samples representing single points in time, focusing upon high titer antibodies at their peak response and defining fractions reacting with epitopes throughout entire domains. We propose to study fractions of the polyclonal responses that are directed to clusters of surface epitopes on factor VIIl's C domains to characterize variability between subjects and stability (or changes) across time in individual patients. We will study three sets of patient groups: (1) allo antibodies in severe hemophilia A, (2) auto antibodies in acquired hemophilia A and (3) combined auto and allo antibodies in patients with mild hemophilia A. C domain epitopes remain the most complex and least well understood. Affinity-purified antibody fractions binding to C domain epitope clusters will be characterized. We have expressed C2 and C1C2 and several mutants. Relative amounts of inhibiting and/or binding patient antibodies reacting to C2, its mutants or C1C2 will be assessed as will distribution of IgG isotypes, both between patients and longitudinally in a given patient's samples. Specific hemophilic C1C2 mutants will characterize responses in mildly severe patients with inhibitors. As a final component, we will define structural elements of C1C2's surface, and the extent of flexibility in conformations of residues on C2, surface residues that differ when the entire C domain is present. Results will provide insights into strategies for therapy of bleeding episodes and the induction of tolerance or prevention of alloimmunization.